1323 Anal. Calcd for C12H14N204: C, 57.59; H, 5.64; N, 11.19. Found: C, 57.76; H, 5.80; N, 11.47. 2-(~-arabino-l’,2’,3’-Triacetoxybutyl)quinoxalhe (Enantiomer of 6c). A suspension of 18.0 g (0.1 mol) of rhamnose hydrate, 11.0 g (0.1 mol) of o-phenylenediamine, 10 ml (0.21 mol) of anhydrous hydrazine, and 6.0 ml (0.1 mol) of acetic acid in 200 ml of pyridine was stirred at 100” for 4 hr. The clear red solution was concentrated in uacuo. The residue was extracted with 200 ml of propanol. The alcoholic extract was decolorized with charcoal and concentrated. Treatment of the oily, amber residue with 120 ml of pyridine-acetic acid, 1 :1, gave 5.37 g of crystalline N,N’diacetyl-@phenylenediamine, which was removed by filtration. Concentration of the filtrate and trituration of the residue with 150 ml of benzene gave an additional 4.63 g (61 total recovery) of N,N’-diacetyl-o-phenylenediamine. The benzene filtrate was extracted three times with 25-ml portions of 6 Nsulfuric acid, extracted with water, and decolorized with charcoal. Removal of the benzene gave 24.3 g of red-brown oil. A portion of the oil (8.03 g) was further purified by elution from
a 250-g column of alumina with carbon tetrachloride-chloroform solvent. One liter of solvent eluted 2.48 g of liquid 2-(~-arabino1’,2’,3’-triacetoxybutyl)quinoxalinewhich was freed of solvent by heating in a rotary evaporator under vacuum until constant pres236 mp (E 31,500), sure readings were obtained; uv spectrum 309.5 mp (shoulder,E 5600),and 317.5 mp (e 6500). Anal. Calcd for C18H20N206: C, 59.99; H, 5.59; N, 7.77. Found: C, 59.91; H, 5.74; N, 7.65. Acknowledgment. This investigation was supported by U. S . Public Health Service Research Grant G M 11966 from the National Institute of General Medical Sciences. The Cary 60 spectropolarimeter was purchased with funds from the Public Health Service grant and from National Science Foundation Grant GP-2125. We thank Mr. Robert Butenko for assistance in preparing some of the compounds.
The Synthesis of Deamino-oxytocin by the Solid Phase Method’ Herbert Takashima,2a V. du Vigneaud,2band R. B. MerrifieldZc
Contribution f r o m the Department of Biochemistry, Cornell University Medical College, New York, New York 10021, the Department of Chemistry, Cornell University, Ithaca, New York 14850, and the Rockefeller University, New York, New York 10021. Received October 4,1967 Abstract: Deamino-oxytocin, a highly potent analog of oxytocin, has been synthesized by the solid phase method. The fully protected polypeptide-nitrated resin compound, S-benzyl-P-mercaptopropionyl-O-benzyl-L-tyrosyl-L-isoleucyl-~-glutaminyl-~-asparaginyl-S-benzyl-~-cysteinyl-~-prolyl-~-leucylglycyl nitrated resin, was prepared and cleavage of the peptide chain from the resin was effected by ammonolysis, Debenzylation of the protected polypeptide followed by oxidative cyclization of the resulting deamino-oxytoceine yielded deamino-oxytocin. The purified deamino-oxytocin crystallized readily from water. This crystalline analog possessed full biological activity.
T
he solid phase method3+ has facilitated the synthesis of peptides since it offers speed and simplicity as well as good yields, We therefore desired to apply this solid phase technique t o the synthesis of analogs of the posterior pituitary hormones oxytocin and vasopressin. To this end the synthesis of deamino-oxyt0cin,~-9 a highly potent crystalline analog of oxytocin, was undertaken. The synthesis of deamino-oxytocin by the resin method essentially followed the procedure outlined previously for the synthesis of angiotensins by this techniquet5 except for the fact that the polymer support was used in the nitrated form3 in order to facilitate the subsequent cleavage of the peptide by ammonolysis. l o (1) This work was supported in part by Grant HE-01675 to Cornell University Medical College and by Grant HE-11680 to Cornell University from the National Heart Institute, U. S. Public Health Service. (2) (a) Cornell University Medical College and Cornell University; (b) Cornell University Medical College and Cornell University; to whom inquiries should be directed at the Department of Chemistry, Cornell University, Ithaca, N. Y. 14850; (c) The Rockefeller University. (3) R. B. Merrifield, J . Amer. Chem. Soc., 85, 2149 (1963). (4) R. B. Merrifield, Biochemisrry, 3, 1385 (1964). ( 5 ) G. R. Marshall and R. B. Merrifield, ibid., 4, 2394 (1965). (6) V. du Vigneaud, G. Winestock, V. V. S. Murti, D. B. Hope, and R. D. Kimbrough, Jr., J . B i d . Chem., 235, PC64 (1960). (7) D. B. Hope, V. V. S. Murti, and V. du Vigneaud, ibid., 237, 1563 (1962). (8) D. Jarvis and V. du Vigneaud, Science, 143, 545 (1964). (9) B. M. Ferrier, D. Jarvis, and V. du Vigneaud, J . B i d . Chem., 240, 4264 (1965).
2-Butyloxycarbonyl (Boc) amino acids were used and N,N’-dicyclohexylcarbodiimide” was the coupling reagent except for the coupling of the asparagine a n d glutamine residues. The latter were incorporated into the growing peptide chain by means of their nitrophenyl estersI2 since nitrile formation has been observed in coupling reactions involving these amino acids when N,N ‘-dicyclohexylcarbodiimide was used as the coupling reagent. l 3 However, once the glutamine and asparagine residues have been incorporated into the peptide chain, carbodiimide may be used for subsequent couplings without danger of nitrile formation. 14,15 Boc-glycine was esterified t o nitrated chloromethylcopolystyrene-2 % divinylbenzene, and the stepwise synthesis was carried through eight cycles5 to give the fully protected polypeptide-nitrated resin compound, S-benzyl-P-mercaptopropionyl-O-benzyl-L-tyrosyl-L-iso1eucyl-L-glutaminyl-L-asparaginyl-S-benzyl-L-cysteinyl-Lprolyl-L-leucylglycyl nitrated resin. In preliminary runs incomplete coupling was encountered in extending the chain beyond the glutamine (10) M. Bodanszky and J. T. Sheehan, Chem. Ind. (London), 1423 (1964). (11) J. C. Sheehan and G. P. Hess, J . Amer. Chem. Soc., 77, 1067 ( 1955 ) . (12) M. Bodanszky and V. du Vigneaud, ibid., 81, 5688 (1959). (13) D. T. Gish, P. G. Katsoyannis, G. P. Hess, and R. J. Stedman, ibid., 78, 5954 (1956). (14) R. Paul and A. S . Kende, ibid., 86, 741 (1964). (15) D. V. Kashelikar and C. Ressler, ibid., 86, 2467 (1964).
Takashima, du Vigneaud, Merrifield 1 Synthesis of Deamino-oxytocin
1324 cooled to 0". The mixture was stirred for 1 hr at 0" and then residue. This problem was circumvented by deblocking poured onto crushed ice. The light tan nitrated resin was filtered with trifluoroacetic acid16r'7 (instead of 1 N HCl in and washed with solutions that changed gradually from water to glacial acetic acid) and by increasing the coupling time. pure dioxane and then progressively to 100% methanol. The The stepwise synthesis of the protected polypeptidenitrated resin was dried in vacuo over KOH pellets; yield, 52.9 g. Boc-glycyl Nitrated Resin. A solution of 0.83 g (4.7 mmol) of nitrated resin compound was then effectively carried Boc-glycine and 0.59 ml (4.7 mmol) of triethylamine in 20 ml of out. ethyl acetate was added t o 5.04 g of nitrated chloromethylcopolyCleavage of the protected peptide chain from the styrene-2 divinylbenzene. The reaction mixture was stirred at nitrated resin was effected by ammonolysis of the benzyl reflux temperature for 24 hr. The esterified resin was filtered off ester linkage by which the peptide is attached to the and washed with ethyl acetate (three times), methanol (three times), water (three times), and again with methanol (three times). The polymer support'O to give the protected polypeptide, S-benzyl-P-mercaptopropionyl-O-benzyl-L-tyrosyl-L-iso-esterified resin was then dried in vacuo over KOH pellets; yield, 5.24 g. Amino acid analysis of an acid hydrolysate (dioxane-12 N leucyl-~-glutaminyl-~-asparaginyl-S-benzyl-~-cysteinyl-~HCI, 1 : 1) showed the product to contain 0.22 mmol of glycine/g prolyl-L-leucylglycinamide. This peptide intermediate of esterified resin. Boc-L-glutaminyl-L-asparaginyl-S-benzyl-L-cysteinyI-L-prolyldiffers from that used in the synthesis of deamino-oxyL-leucylglycyl Nitrated Resin. The Boc-glycyl nitrated resin ( 5 g) tocin by the stepwise nitrophenyl ester m e t h ~ d ~in) ~ , ~ was placed in the reaction vessel. The following cycle of deprotecthat the 0-benzyl protecting group of the tyrosine resition, neutralization, and coupling was carried out for the introducdue is still intact and has not been cleaved during the tion of each new residue: (1) three washings with 25-ml portions of deprotection step. glacial acetic acid; (2) cleavage of the Boc group by treatment with 1 Ri HC1 in glacial acetic acid (25 ml) for 30 min at 25'; (3) three Debenzylation of the protected polypeptide was washings with 25-ml portions of glacial acetic acid; (4) three washperformed with sodium and liquid ammonia by the ings with 25-ml portions of absolute ethanol; (5) three washings method of Sifferd and du Vigneaud'B as used in the with 25-ml portions of dimethylformamide; ( 6 ) neutralization of synthesis of oxytocin, '9 and oxidative cyclization of the the hydrochloride with 3 ml of triethylamine in 25 mi of dimethylformamide for 10 min; (7) three washings with 25-ml portions of resulting deamho-oxytoceine with potassium ferridimethylformamide; (8) three washings with 25-ml portions of cyanide yielded deamino-oxyt~cin.~ The deaminomethylene chloride; (9) addition of 3.3 mmoles of the appropriate oxytocin was purified by partition chromatography on Boc-amino acid in 20 ml of methylene chloride and mixing for 10 Sephadex G-25*O in the solvent system l-butanolmin; (10) addition of 3.3 mmol of N,N'-dicyclohexylcarbodiimide benzene-3.5 % aqueous acetic acid (containing 1.5 in 5 ml of methylene chloride, followed by a reaction period of 3 hr at 25'; (11) three washings with 25-ml portions of methylene pyridine) (1 : 1 : 2).9 The Rr of the deamino-oxytocin chloride; (12) three washings with 25-ml portions of absolute was identical with that of deamino-oxytocin prepared ethanol. by the stepwise nitrophenyl ester m e t h ~ d . ~The lyophThe coupling reactions involving Boc-L-glutamine and BOC-Lilized powder from partition chromatography was asparagine were carried out riu their nitrophenyl esters. After steps 1-7 of the cycle were carried out, a solution of 3.3 mmol of the subjected t o gel filtration on Sephadex G-25 (200-270 p-nitrophenyl ester of the appropriate Boc-amino acid in 15 mi of mesh)*' equilibrated with 0.2 N acetic acid. This freshly distilled dimethylformamide was added, and the reaction purified deamino-oxytocin prepared by the solid phase was allowed to proceed overnight. The polypeptide-nitrated resin method crystallized readily from watersg The crystalwas washed three times with 25-ml portions of dimethylformamide line analog possessed within the limits of experimental followed by three washings with 25-ml portions of absolute ethanol and drying in vacuo over KOH pellets; yield, 5.89 g (0.2 mmol of error the avian vasodepressor activity of 975 units/mg peptide/@. reported by Ferrier, Jarvis, and du V i g n e a ~ d . ~ Boc-L-isoleucyl-L-glutaminyl-L-asparaginyl-S-benzyl-L-cys teinylThe solid phase method has thus been successfully L-prolyl-L-leucylglycyl Nitrated Resin. The Boc-L-glutaminyl-Lapplied to the synthesis of deamino-oxytocin, a highly asparaginyl-S-benzyl-L-cysteinyl-Lprolyl-L- leucylglycyl nitrated resin (3.5 g, equivalent to 0.7 mmol) was placed in the reaction flask. potent analog of the posterior pituitary hormone Steps 1-12 of the reaction cycle were carried out with the following oxytocin. The deamino-oxytocin prepared by this modifications and with the use of 15-ml portions of solvents in place method was obtained in good over-all yields and posof the 25-ml portions used in the prior couplings: step 2, cleavage sessed full biological activity. It should be mentioned of the Boc group of the glutamine residue by treatment with 15 ml that Beyerman22has carried out the solid phase synof trifluoroacetic acid for 15 min at 25"; step 6, neutralization of the trifluoroacetate with 0.6 ml of triethylamine in 15 ml of dimethylthesis of the partially protected nonapeptide leading t o formamide for 5 min; step 9, addition of 0.45 g (1.98 mmol) of 9-deamido-oxytocinZ3 using the active ester coupling Boc-L-isoleucine in 12 ml of methylene chloride; step 10, addition method with 1,2,4-triazole catalysis, and Manningz4 of 0.42 g (1.98 mmol) of N,N'-dicyclohexylcarbodiimide in 3 ml of has utilized the solid phase technique for the synthesis methylene chloride, followed by a reaction period of 16 hr. S-Benzyl-P-mercaptopropionyl-O-benzyl-L-tyrosyl-L-isoleucylof oxytocin.
z
Nitrated Chloromethylcopolystyren~2~ Divinylben~ene.~Dry chloromethylated copolystyrene-2 divinylbenzene (40 g) (BioBeads S.X-2, 2 W 4 0 0 mesh, capacity 1.5 mequivlg) was added slowly with stirring t o 400 ml of fuming nitric acid that had been (16) H. Kappeler and R. Schwyzer, Helu. Chim. Acta, 43, 1453 (1960). (17) D. G. Smyth, W. H. Stein, and S . Moore, J . B i d . Chem., 237, 1845 (1962). (18) R. H. Sifferd and V. du Vigneaud, ibid., 108, 753 (1935). (19) V. du Vigneaud, C. Ressler, J. M. Swan, C. W. Roberts, and P. G. Katsoyannis, J . Amer. Chem. SOC.,76, 3115 (1954). (20) D. Yamashiro, Nature, 201, 76 (1964). (21) J. Porath and P. Flodin, ibid., 183, 1657 (1959). (22) Personal communication from Professor H. C. Beyetman. (23) B. M. Ferrier and V. du Vigneaud, J . Med. Chem., 9, 55 (1966). (24) Personal communication from Dr. Maurice Manning.
Journal of the American Chemical Society
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glutaminyl-L -asparaginyl-S benzyl L cysteinyl-L-prolyl-L-leucylglycyl Nitrated Resin. The 0-benzyl-L-tyrosine and S-benzyl-Pmercaptopropionic acid residues were incorporated onto the BOC-Lisoleucyl- L-glutaminyl -L-asparaginyl- S-benzyl-L-cysteinyl-L-prolylL-le.ucylglycy1nitrated resin utilizing steps 1-12 of the reaction cycle previously described and using a 15-ml portion of the appropriate solvent for each washing. Solutions of 1.98 mmol of the protected amino acid in 12 ml of methylene chloride and of 1.98 mmol of N,N'-cyclohexylcarbodiimide in 3 ml of methylene chloride were used in steps 9 and 10, respectively. The coupling reactions were allowed to proceed overnight. After step 12 following the incorporation of the S-benzyl-P-mercaptopropionic acid, the protected polypeptide-nitrated resin compound was further washed with 15-ml portions of glacial acetic acid (three times), absolute ethanol (three times), and methylene chloride (three times). The product was dried iri cacuo over KOH pellets overnight. S-Benzyl-P-mercaptopropionyl-O-benzyl-L-tyrosyl-L-isoleucylL-glutaminyl-L-asparaginyl- S-benzyl-L-cysteinyl-L-prolyl-L-1eucylL
Experimental Section
1 90:5 ] February 28, 1968
1325 (containing 1.5 pyridine) (1 :1 :2) and applied to a Sephadex G-25 glycinamide. Dry ammonia was bubbled into a stirred suspension of 3.54 g of the S-benzyl-P-mercaptopropionyl-0-benzyl-L-tyrosyl- (10@-200 mesh) column (2.16 X 110 cm) that had been equilibrated with the lower phase. The column was eluted with the upper phase L isoleucyl-r. glutaminyl-r. asparaginyl-S-benzyl-L-cysteinyl-L-proand 96 fractions of 9.5 ml each were collected. The chromatogram lyl-L-leucylglycylnitrated resin in 150 ml of anhydrous methanol at obtained by plotting the Folin-Lowry color valuesz6of the fractions 0" for 1.5-2 hr (until the solution was saturated with ammonia). showed a single peak with a maximum at fraction 52 (Rr 0.19; The reaction mixture was stirred overnight at 0-4°.26 The methlit.9 0.19). The fractions corresponding to this peak were pooled, anol and ammonia were removed under aspirator vacuum. Ditwice the volume of water was added, and the resulting mixture methylformamide (200 ml) was added to the dry residue and the was concentrated under reduced pressure and lyophilized; yield, suspension was stirred vigorously for 3 hr. The resin was filtered off and washed three times with dimethylformamide. 49 mg (41 The lyophilized powder was dissolved in 6 ml of 0.2 N acetic acid The solvent was removed from the combined filtrate and washings and subjected to gel filtrationz1on a Sephadex G-25 (200-270 mesh) on a rotary evaporator at below 40". The residue was dissolved in column (2.82 x 64 cm) that had been equilibrated with 0.2 N 20 ml of dimethylformamide and 15 ml of distilled water was added acetic acid. The column was eluted with 0.2 N acetic acid and 120 gradually with stirring. The turbid solution was allowed to stand fractions of 4.9 ml each were collected. A plot of the Folin-Lowry in the refrigerator overnight. The precipitate was filtered off, color values of the various fractions showed a single symmetrical washed with ethanol, and dried in cucuo over KOH pellets; yield, peak with a maximum at fraction 65. The fractions corresponding 197 mg, mp 234236". to this peak were pooled and lyophilized to give a white powder; A sample was prepared for analysis by reprecipitating twice from ~ (c 0.5, 1 N acetic acid), lit. [ ~ ] " O D yield, 39.4 mg; [ a I z 5-95.1" a dimethylformamide-water mixture. The compound was filtered -88.3" ( c 0.5, 1 N acetic acid)g and [ a I z 1-107" ~ (c 0.5, 1 Nacetic off, washed with ethanol, and dried in L'UCUO, over KOH pellets; acid).' D 37.3" (c 0.6, dimethylformamide). mp 238-240", [ c Y ] ~ ~This lyophilized powder (25 mg) was dissolved in 0.8 ml of water Anal. Calcd for C84H15012NllS2:C, 60.8; H, 6.78; N, 12.2. in a water bath at 80-90". The solution was filtered through a Found: C,60.9; H,6.78; N, 11.9. Deamino-oxytocin. S-Benzyl-P-mercaptopropionyl-0-benzyl- sintered-glass funnel and was allowed to stand at room temperature for 5 hr. Crystals began to form within 1 hr and the bulk of the L tyrosyl - L-isoleucyl L glutaminyl L - asparaginyl- S-benzyl-L-cysdeamino-oxytocin had crystallized out after 5 hr. The solution was teinyl-L-prolyl-L-leucylglycinamide (155 mg) was dissolved in 125 allowed to stand overnight in the refrigerator. The deaminoml of stirred boiling liquid ammonia (distilled from sodium in a n oxytocin crystals were filtered off, washed with water, and dried all-glass apparatus). A fresh sodium stick was introduced and within ~ a c u o ;yield, 14.8 mg; mp 182-183', uncorrected (Fisher-Johns drawn when the blue color pervaded the solution. The sodium melting point apparatus; electrically heated aluminum block), stick was momentarily introduced intermittently until the blue color lit.9 mp 179" (modified Kofler block, corrected) and 178-184" persisted for 30 sec. The ammonia was removed by lyophilization (capillary, corrected); [aIz1D -103.3' ( c 0.46, 1 N acetic acid), at the water pump and the residue was dissolved in 300 ml of 0.03 ~ (c 0.5, 1 N acetic acid). lit.9 [ a I z 0-90.4' trifluoroacetic acid. The p H of the solution was adjusted to 8.0 with ammonium hydroxide and an excess of 0.1 N potassium ferricyanide (4.5 ml) was added to the stirred solution. After 15 min Acknowledgments. The authors are indebted t o the AG 3-X4 resin (chloride form) was added and stirring was confollowing: Mr. Joseph Albert for the elemental analytinued for 15 min to remove ferrocyanide and excess ferricyanide ses; Mr. Roger Sebbane for the amino acid analyses; ions. The resin was removed by filtration, and the solution was and Miss Margitta Wahrenburg and Mrs. Jessie Lawlyophilized. The lyophilized residue was dissolved in 20 ml of the upper phase rence for the bioassays, under the direction of Dr. W. aqueous acetic acid of the solvent system 1-butanol-benzene-3.5 Y. Chan.
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z).
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( 2 5 ) Ammonolysis conditions as described to us by Dr. Maurice Manning in a personal communication.
(26) 0. H.Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. B i d . Chem., 193, 265 (1951).
The Structure of Frenolicin' George A. Ellestad, Martin P. Kunstmann, Howard A. Whaley, and Ernest L. Patterson
Contribution f r o m Lederle Laboratories, A Division of American Cyanamid Company, Pearl River, New York 10965. Received September 14, 1967 Abstract: Evidence is put forward which describes the structure and stereochemistry of frenolicin as I.
T
he isolation and preliminary characterization of frenolicin, a pale yellow crystalline antibiotic from Streptomyces fradiae, were described by Van Meter, Dann, and Bohonos.* They suggested the molecular formula t o be CI3Hl4Ojand indicated the presence of two acidic functions assigned t o a phenolic or enolic grouping and a carboxylic acid. In addition, they (1) A portion of this work was reported in preliminary form: G. A. Ellestad, H. A. Whaley, and E. L. Patterson, J . Am. Chem. SOC.,88, 4109 (1966). (2) J. C..Van Meter, M. Dann, and N. Bohonos, "Antibacterial Agents Annual-1960,'' Plenum Press, New York, N. Y.,1961, p 77.
observed that frenolicin readily absorbed 2 mol of hydrogen and contained a C-methyl group. We wish to report in detail work which has led to the structure and relative stereochemistry of frenolicin as that formulated by the novel naphthoquinone epoxide La Repetition of the elemental analyses gave figures more indicative of the formula C18H1807,rather than that previously proposed, and the molecular weight (346) required by this formula was confirmed by mass spec(3) Symbols a' and e' denote pseudo-axial and pseudo-equatorial configurations of the bonds in question.
Effestad,Kunstrnann, Whaley, Patterson 1 The Structure of FrenoIicin
